Squamous cell carcinoma of the head and neck (HNSCC) is the 6th most common cancer in the developed world, affecting nearly 44,000 patients each year in the US, which results in ~11,000 deaths. The vast majority of these malignancies involve neoplastic lesions in the oral cavity, lip, and pharynx. Like most cancers, HNSCC results from a series of discrete, irreversible and sequential alterations in genes that control cell growth and differentiation, together with genetic aberrations promoting invasion and metastasis. The goal of our program is scientific excellence in addressing the devastating problem of oral cancer. In particular, we aim to elucidate the molecular changes that contribute to the evolution of oral neoplasia, and to use this knowledge to develop markers of disease progression and novel therapeutic approaches for oral malignancies. Gene discovery and gene expression evaluation and in HNSCC. The completion of the human genome project and the recent development of novel, highly sensitive high-throughput techniques have now afforded the unique opportunity to perform a comprehensive molecular characterization of normal, precancerous, and malignant cells. As part of these efforts, a joint program was established between NCI and NIDCR aimed to identify the nature of those genes expressed during HNSCC development. Analysis of the database of cDNA libraries derived from microdissected normal and HNSCC tissues contributed by our lab with bioinformatic tools identified a large number of potential novel genes. Their differential expression in normal and tumor tissues was examined using an oral cancer specific cDNA microarray. Known genes that are highly expressed in normal or HNSCC libraries were also identified. Furthermore, using laser capture microdissection and recently developed linear amplification techniques, we are now examining gene expression patterns in normal and tumor tissues, focusing on SCC from the tongue, a major oral cancer site, and carcinomas of the nasopharynx, an etiologically distinct HNSCC. In parallel, efforts are currently underway to use serial analysis of gene expression (SAGE) to catalog HNSCC transcribe genes (HNSCC transcriptome). These efforts, together with other multi-institutional initiatives, are likely to contribute to the complete understanding of the molecular pathogenesis of HNSCCs, thus helping to identify new markers for the early detection of preneoplastic lesions and novel targets for pharmacological intervention. Persistent activation of the Akt pathway in HNSCC: A potential target for UCN-01, which displays potent antitumor activity in this cancer type. As part of ongoing efforts to unravel the molecular events underlying tumor progression in HNSCC, we have explored the nature of the proliferative pathways whose dysregulation may promote aberrant cell growth in this tumor type. The serine/threonine protein kinase Akt is a key regulator of normal and cancerous growth and cell fate decisions. In this regard, using a mouse model for chemically-induced squamous carcinoma (SCC), we have recently observed that elevation in the kinase activity of Akt represents an early event during papilloma formation, which precedes SCC. Prompted by this observation, we have examined the status of activation of Akt in different stages of SCC development in mice and in clinical samples from HNSCC patients. By immunohistochemical analysis using phosphorylation-state specific antibodies, we have demonstrated that Akt activation correlates closely with the progression of mouse skin SCC, and observed that activation of Akt is a frequent event in human HNSCC. Indeed, active Akt can be detected in these tumors, and its level of expression and nuclear localization correlated with the severity of the cancer lesions. On the other hand, our team has continued its drug evaluation effort aimed to developing novel therapies for improving the quality of life and life expectancy of HNSCC patients. In this regard, UCN-01 (7-hydroxy-staurosporine), a protein kinase C and cdk modulator that exhibits antiproliferative and antitumor properties in some experimental tumor models, represented a potential candidate to test in HNSCC. In collaboration with the Developmental Therapeutic Program (DTP), NCI, and the Molecular Therapeutic Unit, we found that UCN-01 displays potent antiproliferative properties in vitro in HNSCC cells and in vivo in HNSCC xenografts. Total sustained abolition of tumor growth (p<0.00001) was obtained with only one cycle of UCN-01 treatment for 5 consecutive days, thus together suggesting that UCN-01 may represent a suitable drug candidate for treating HNSCC patients. Of interest, we have recently obtained evidence that the Akt signaling pathway may represent a biologically relevant target for UCN-01, at concentrations safely achievable in clinically relevant situations. Constitutive activation of STAT3 in HNSCC: Evidence for an EGFR-independent pathway mediated by the autocrine/paracrine stimulation of the IL6/gp130 cytokine system. The aberrant growth of HNSCC is often associated with the constitutive activation of signal-transducer-and-activator-of-transcription-3 (STAT3), which has been correlated with the persistent stimulation of EGF receptors that are highly expressed in HNSCC. To investigate the mechanism underlying STAT3 deregulation in HNSCC, we examined the interplay of the STAT3 and EGFR signaling pathways using a panel of HNSCC cell lines. We found that while STAT3 was active in most cell lines, only few (3 out of 10) of them were moderately to strongly positive for activated EGFR, and that even in the EGFR-positive cell lines, STAT3 activation was not dependent on EGFR activation. Interestingly, we found that conditioned medium harvested from HNSCC cells could induce STAT3 tyrosine phosphorylation in keratinocyte lines independent of EGFR signaling. Instead, we found that interleukin-6 (IL6) as the major autocrine/paracrine factor for STAT3 activation, which coincided with high levels of secretion of IL6 into the culture medium by these cancer cells. Furthermore, we obtained evidence that constitutive activation of STAT3 in HNSCC may utilize an autocrine/paracrine-activating loop mediated by IL6 and other cytokines acting through the gp130 receptor family, which may confer both proliferative and survival potential in this malignancy. Development of conditional animal models for squamous carcinogenesis: With the assistance of our NIDCR Gene-targeting Core Facility, we have generated transgenic mice carrying the tetracycline-inducible system (tet-on receptor) targeted to the basal layer of stratified epithelium using the cytokeratin 5 promoter. Transgenes of interest, including candidate oncogenes, will be expressed under the control of a tetracycline-responsive promoter. As a proof of principle, we have examined the ability of active ras genes to promote SCC when conditionally expressed. Of interest, although prior animal models suggested that active ras alone promotes benign papilloma formation with a variable rate of delayed carcinoma conversion, the use of this system, which targets a cell compartment that includes the epithelial stem cells, was sufficient to promote the rapid formation of SCC in the skin and oral tissues of animals treated with doxycycline. Using this model, we are now positioned to test the transforming potential in vivo of activated alleles of newly discovered signaling molecules, alone or in animals engineered to be deficient in relevant tumor suppressor genes. Current and future use of this model system may help to unravel the mechanisms responsible for SCC, and aid in the search for alternative oral cancer treatments.